RNA polymerase II (pol II) transcription through a nucleosome is one of the most fundamental processes in eukaryotic gene expression. Genetics, chromatin immunoprecipitation and molecular studies in the yeast S. cerevisiae have provided many insights into pol II elongation in vivo. Despite the extensive literature there is a poor understanding of the actual mechanism because no lab has attacked the issue in a systematic manner using in vitro elongation systems with chromatin templates. The phenomena of co-transcriptional histone acetylation and rapid deacetylation, and the related process of co-transcriptional methylation and demethylation are of particular interest due to their conservation in all eukaryotes. Yet even a rudimentary understanding of the mechanism requires that these events be recreated and analyzed in a defined transcription system with purified proteins. I recently completed a 1-year sabbatical in Jerry Workman's lab at the Stowers Institute, where I set up an in vitro transcription elongation system on mononucleosomes. I found that acetylation and ATP-dependent remodeling work in concert to permit pol II passage through the histone octamer. I propose to significantly extend these studies to address several aspects of the elongation mechanism. My group will isolate relevant yeast proteins using tandem affinity purification and employ these in chromatin binding and transcription assays. A particularly powerful technique that will form the cornerstone of the proposal is the immobilized template assay. This assay uses biotinylated, chromatinized templates attached to streptavidin-coated beads to capture the elongation complex. The complexes will then be subjected to functional and compositional analyses. This technique and more traditional methods such as electrophoretic mobility shift will be used to address the three aims below.
Aim #1 will examine the mechanism by which pol II passes through a nucleosome and the fate of the histone octamer on mono and poly-nucleosomal templates.
This aim will utilize purified histone acetyltransferases (SAGA and NuA4) and ATP dependent remodeling machines (RSC and SWI/SNF).
Aim #2 will explore current models for the function of H3K4 and H3K36 trimethylation and the detailed mechanisms of the machines that recognize the methylated histones including SAGA, Rpd3S and Chd1.
Aim #3 will examine how histone chaperones, including Spt6, Asf1 and FACT, assemble and disassemble nucleosomes with an emphasis on how covalent modifications affect the process. Our study will leverage the vast body of knowledge from yeast genetics and molecular biology to craft and test hypotheses for how pol II passes through a nucleosomes and how covalent modifications of chromatin regulate this process. The knowledge will provide fundamental information applicable to pol II elongation in all eukaryotes. B. Project Narrative One of the most important aspects of gene regulation is understanding how chromatin, the nucleoprotein structure that protects eukaryotic genome, is removed when genes are turned on and replaced when genes are turned off. This step is fundamental to all organisms and knowledge of the process is key to understanding gene regulation during disease, differentiation and development in humans. Our proposal will use the yeast S. cerevisiae as a model organism to understand this process.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics A Study Section (MGA)
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Carter, Anthony D
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University of California Los Angeles
Schools of Medicine
Los Angeles
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Lin, Justin J; Carey, Michael (2012) In vitro transcription and immobilized template analysis of preinitiation complexes. Curr Protoc Mol Biol Chapter 12:Unit 12.14.